The Bacillus genus comprises numerous endospore-forming bacteria that have myriad uses in the agricultural and animal nutrition fields, among others. Several strains of Bacillus are currently marketed for use as plant growth promoters and/or biocontrol agents against insect pests and diseases (see, e.g., Masaaki Morikawa, “Beneficial Biofilm Formation by Industrial Bacteria Bacillus subtilis and Related Species,” Journal of Bioscience and Bioengineering (2006) 101(1):1-8; Kloepper, et al., “Induced Systemic Resistance and Promotion of Plant Growth by Bacillus spp.,” Phytopathology (2004) 94(11):1259-1266). These organic, environmentally-friendly alternatives have found wide-spread acceptance among agronomists and horticulturists due to their effectiveness as plant growth promoters and as biopesticides.
Bacillus subtilis is a Gram-positive soil bacterium, which is often found in the plant rhizosphere. B. subtilis, like many species of bacteria, can exhibit two distinct modes of growth, a free-swimming, planktonic mode of growth and a sessile biofilm mode in which an aggregate of cells secrete an extracellular matrix to adhere to each other and/or to a surface (Branda, et al., “Fruiting Body Formation by Bacillus subtilis,” Proc. Natl. Acad. Sci. USA (2001) 98:11621-11626; Hamon and Lazazzera, “The Sporulation Transcription Factor Spo0A is Required for Biofilm Development in Bacillus subtilis,” Mol. Microbiol. (2001) 52:847-860). The pathways utilized by bacteria such as B. subtilis to build biofilms are extremely diverse, varying enormously within and among different species and under different environment conditions (Bais, et al., “Biocontrol of Bacillus subtilis Against Infection of Arabidopsis Roots by Pseudomonas syringae is Facilitated by Biofilm Formation and Surfactin Production,” Plant Physiol. (2004) 134:307-319; Lemon et al., “Biofilm Development with an Emphasis on Bacillus subtilis,” (2008) Current Topics in Microbiology and Immunology (2008) 322:1-16). It has somewhat recently been recognized that biofilm formation by specific strains of B. subtilis and related species may help control infection caused by plant pathogens (Morikawa (2006), supra).
Biofilm morphology and chemical composition vary across species and strains. Mucoid colony morphology and production of γ-polyglutamic acid occurring in wild Bacillus subtilis strains has been correlated with enhanced biofilm formation, while flat, dry colony morphology occurring in domestic (or lab) strains has been correlated with decreased biofilm formation. See Stanley, N. and Lazazzera, B. “Defining the Genetic Differences Between Wild and Domestic Strains of Bacillus subtilis that Affect Poly-γ-DL-Glutamic Acid Production and Biofilm Formation,” Molecular Microbiology (2005) 57(4): 1143-1158 at 1145. The Branda paper (supra, 2001) described deficiencies in biofilms with non-mucoid colony morphology, such as flat, small, dry colonies, which grew laterally and eventually fused with each other, leading to small colonies lacking aerial structures. The Stanley paper, however, described a hybrid Bacillus subtilis strain having a loss of function mutation in the swrA locus that formed flat, dry colonies and showed enhanced biofilm formation. (The hybrid strain was a congression-made combination of a domestic strain with the DNA from a wild strain that is responsible for mucoid colony morphology.) Applicants have found that wild Bacillus strains with reduction or loss of function mutations to the swrA locus produce flat, dry colonies that form robust biofilms and, further, that formulated fermentation products consisting of such cells enhance plant health, lead to more robust root colonization compared to strains containing the wild type swrA gene, and control plant diseases and pests, such as nematodes.
Commercial agriculture and home gardening would both benefit from the availability of different and improved sources of Bacillus strains for use in enhancing plant growth, promoting plant health, controlling plant pests and diseases and providing alternatives to chemical nematicides. The present invention provides a new class of such bacterial strains and improved methods of their use by manipulation of biofilm formation.